Immunstimulating lipid formulation

ABSTRACT

A pharmaceutical formulation for parenteral or mucosal administration of antigens and/or vaccines to humans and animals, comprising monglyceride preparations having at least 80% monoglyceride content and where the acyl group contains from 6 to 24 carbon atoms, together with fatty acids where the number of carbon atoms may be varied between 4 and 22.

The present invention relates to a novel pharmaceutical formulation foradministration of antigens and/or vaccines. The preferred route ofadministration is via the mucosal membranes, however parenteraladministration may also be used. The invention also relates to the useof certain compounds (as defined below) as adjuvants or vehicles in suchformulation.

BACKGROUND

An increasing number of specific antigens from different types oforganisms (e.g. tumor cells, bacteria, virus and parasites) has beenproduced using cloning techniques over the last years. However, theseantigens are frequently weak immunogens despite their high specificity.

To obtain good protection after vaccination, immune stimulating systemsare needed that can enhance and activate the immune system against theseweak antigens. Such immune stimulating systems are called adjuvants.

Adjuvants, presently mainly used in animal experiments, includes ahighly heterogeneous group of substances; inorganic substances, oilemulsions, charged polymers, neutral substances or substances frombacteria.

There are presently large efforts in research and development in orderto obtain a safe adjuvant with high efficacy to be used in humans.However, today there is presently no general adjuvant for this purpose.

Alum hydroxides and alum phosphates were the first two inorganicsubstances that were used in humans. The immune response obtained is aresult of slow desorption of the precipitated antigen on the surface ofthe particle. Later it was shown that phagocyting cells were attractedby these alum salts leading to further enhancement of the immuneresponse. However, these salts are not safe since granuloma formationhas been reported (Slater et al, Br. J. Dermatol. (1982) Vol. 107, page.103-108.). Furthermore, the alum salts can not be used for all antigenssince all antigens are not adsorb on the surface.

In 1944 Freund introduced his adjuvant consisting of a mixture ofvegetable oil, mineral oil, detergents and killed bacteria. Theenhancement obtained was partly due to slow release of the antigen fromthe oil emulsion. Freunds adjuvant can however not be used in humans dueto granuloma formation, induction of auto-immune reactions and thenon-biodegradable mineral oil. Furthermore, the effect is difficult tocontrol. The active substance in Freunds adjuvant has been isolated andits structure determined and shown to be N-acetylmuramyl-L-alaninisoglutamate, often called muramyl-dipeptide (MDP).

The adjuvant effect dependent of the particle size of polymetacrylateand polystyrene particles was examined on mice (Kreuter et al, Vaccine,(1986) vol 4, 125-129) by the use of ovalbumin (adsorbed on theparticles) as a model antigen with subsequent assay of the immuneresponse. The size of the particles was varied between 62 and 306 nm.The result was that smaller particles enhanced the immune responsebetter than larger. The smaller particles gave a better effect than 0.2%Al(OH)₃. All preparations elicited a higher response as comparedto-fluid preparations. Similar experiments where particulate systemswith smaller size results in a higher immune response as compared tolarger particles are known in the scientific literature.

Almost all systems used today for enhancement of the immune responseagainst antigens are particles or is forming particles together with theantigen. In the book “Vaccine Design—the subunit and adjuvant approach”(Ed: Powell & Newman, Plenum Press, 1995) all known adjuvants aredescribed both regarding their immunological activity as well asregarding their chemical characteristics. As described in the book morethan 80% of the adjuvants tested today are particles or polymers thattogether with the antigens (in most cases proteins) are formingparticles. The type of adjuvants that not are forming particles are agroup of substances that are acting as immunological signal substancesand which under normal conditions consists of the substances that areformed by the immune system as a consequence of the immunologicalactivation after administration of particulate adjuvant systems.

Using particulate systems as adjuvants, the antigens are associated ormixed with or to a matrix which has the characteristics of being slowlybiodegradable. Of great, importance using such matrix systems are thatthe matrix does not form toxic metabolites. Choosing from this point ofview, the main-kind of matrices that can be used are mainly substancesoriginating from a body. With this background there are only a fewsystems available that fulfils these demands: lactic acid polymers,poly-amino acids (proteins), carbohydrates, lipids and biocompatiblepolymers with low toxicity. Combinations of these groups of substancesoriginating from a body or combinations of substances originating from abody and biocompatible polymers can also be used. Lipids are thepreferred substances since they display structures that make thembiodegradable as well as the fact that they are the most important partin all biological membranes.

Lipids are characterized as polar or non-polar. The lipids that are ofmost importance in the present invention are the polar lipids since theyhave the capacity to form particulate systems in water. Another way ofdefining these lipids are as amphifilic due to their chemical structurewith one hydrophobic and one hydrophilic part in the molecule therebybeing useable as surface active substances. Examples of main groups ofpolar lipids are mono-glycerides, fatty acids, phospholipids andglycosphingolipids. These main groups can be further characterizeddepending on the length of the acyl chain and the degree of saturationof the acyl chain. Since the number of carbon atoms in the acyl chaincan be in the range of 6 to 24 and the number of unsaturated bonds canbe varied there are an almost infinite number of combinations regardingthe chemical composition of the lipid.

Particulate lipid systems can be further divided into the differentgroups as discussed in the scientific literature such as liposomes,emulsions, cubosomes, cochleates, micelles and the like.

In a number of systems the lipids may spontaneously form or can beforced to form, stabile systems. However, under certain circumstancesother surface active substances has to be introduced in order to achievestability. Such surface active systems can be of non-lipid character butpossess the characteristics of the polar lipids having hydrophobic andhydrophilic parts in their molecular structure.

Another factor that has been shown to be of importance is that lipidsexhibit different physical chemical phases, these phases has indifferent test systems been shown to enhance uptake of biologicalsubstances after administration to mucosal membranes.

In the classical immunology and in combination with vaccination againstdifferent types of infectious agents e.g. bacteria, virus or parasitesthe prevailing dogma has been to administrate the vaccine subcutanouslyor intramuscularly. However, research has during the last years shownthat the body has a very effective immunological system that resides inthe mucosa. It has been shown that you can administrate vaccines orally,nasally, rectally and vaginally. In the same way as for the classicalimmunization it has been shown that by mucosal vaccination there is alsoa need for enhancement of the immunological response by the addition ofadjuvants.

In the same wax, as within the classical immunology where vaccines(antigens) are administrated parenterally, there is within mucosalimmunization a great interest in directing the immunological responsetowards development of humoral and/or cellular response. If you obtain ahumoral response it would be important to direct the response in a waythat a certain class of antibodies would be obtained. In order to obtainsuch a goal, specific immune stimulating agents can be added to theformulation of antigens and adjuvants.

Different types of immune stimulating substances are available. One typeis represented by proteins e.g. PHA, Con A, SEA or different types ofinterferons or interleukines. Another type of substance is representedby MDP, as mentioned above. Additional groups can be characterized aslipid derivatives since they show molecular structures which areamphiphilic. One example of such a substance is called MPL. Anothersimilar substance is Quil A. A number of substances that can beclassified within these categories are described in the book “VaccineDesign—the subunit and adjuvant approach” as discussed above.

It would be extremely valuable to be able to make the immunizationprocedures more effective directing the immunological response towards acertain class or subclass of antibodies and/or to be able to induce astrong T-cell response against the antigens.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found that parenteral or mucosaladministration of a pharmaceutical formulation containing one or two ofthe following adjuvants with admixed antigens and/or vaccines improvesthe immune response against the admixed antigens/vaccines. Saidpharmaceutical formulation for parenteral or mucosal administration ofantigens and/or vaccines to an animal comprise one or more substancesselected from

a) monoglycerides of the general formula

 wherein R₁ and R₂ is H and R₃ is one acyl group containing from 6 to 24carbon atoms, preferably 8 to 20 carbon atoms, even more preferably14-20 carbon atoms and where the acyl chain may contain unsaturatedbonds. In a monoglyceride the acyl chain is normally in the R₁ or R₂position. However there is normally a acyl migration between the 1 and 2carbons in the glycerol molecule resulting in approximately 90% is inthe R₁ position and 10% in the R₂ position. Thus, in the presentinvention distilled 1-monoglycerides from Danisco Ingredients (Denmark)with a purity of more than 80% preferably more than 90%, more preferablyover 95% is used. The diglyceride content is maximum 3% andtriglycerides and fatty acid content is less than 1.0%. Themonoglycerides according to the invention normally contains more thanmore than 80% of a specific fatty acid, preferably over 90%.

and

b) fatty acids of the general formulaCH₃—(CH₂)_(n)—COOH

 where “n” may be varied between 4 and 22, preferably 8 to 18 and wherethe acyl chain may contain one or more unsaturated bonds.

The formulation according to the invention may comprise additionalpharmaceutical excipients selected from the one or several of thefollowing groups; preservatives and osmotic pressure controlling agents,pH-controlling agents, organic solvents, hydrophobic agents, enzymeinhibitors, water absorbing polymers, surfactants and absorptionpromoters, anti-oxidative agents, and the like.

The formulation according to the invention may comprise any antigenand/or vaccine selected among all the antigen and/or vaccines relevantto humans or animals, including marine animals.

This invention discuss lipids which, when mixed with antigens, enhancethe immune activity against the antigens thereby functioning as anadjuvant in various vaccine formulations. Especially the inventioncomprise the use of a formulation for vaccination of the mucosa whichcan be immunologically activated by nasal, oral, vaginal or rectaladministration. The invention also comprise the use of the lipid systemfor parenteral administration. The use of an adjuvant such as describedin the present invention, which can be used both for parenteral as wellas for mucosal administration is not limited to humans. Equallyimportant is the use within the veterinary field for the immunization ofe.g. cattle, pigs, chickens and the like. Furthermore, there is a largeand growing interest in applying both parenteral as well as mucosalvaccines in the field of fish farming. In this area the administrationcan be performed by incorporation of the formulation in the food.Furthermore, the fish may be allowed to swim for a limited period oftime in the vaccine formulation containing the antigens and theadjuvants thus being immunized by the mucosal route via the gills.

In the scientific literature there are reports showing how to enhancethe uptake of a biologically active substance after administration tothe mucosa together with certain lipids. As an example Li & Mirra(Pharm. Res. vol 13:1, 1996) describes the administration of insulinmixed with phospholipids in the form of liposomes to the lung. They showthat the effect is dependent on the length of the acyl chain and thecharge of the particle. Optimal length was 10 carbon atoms and thecharge preferably positive. Even negatively charged particles wereeffective but neutral system were inferior.

In the same way de Haan et al (Vaccine, 13:2, 155-62, 1995) describes amixture of liposomes and the antigen hemeagglutinin. The mixture wasadministrated nasally to rats whereafter a positive immunologicalresponse could be detected. Gupta et al (Vaccine, 14:3, 219-25, 1995)describes that a mixture of diphtheria toxoid together with anon-phospholipid based liposom system administrated parenterally torabbits results in an immune response which was at the same level as themarketed product which was Alum-adsorbed diphtheria toxoid.

A number of scientific reports also show that good immunologicalresponses are obtained after administration of liposomes to the mucosawhere the antigen is entrapped or adsorbed to liposomes.

Studies in vitro on a human cell line obtained from a colon cancer(Caco-2) shows that the best penetrating effect, tested with the modelsubstance mannitol, can be seen with a chain length of 10 carbon atoms.In this case the lipids consisted of the salts of fatty acids. Theobtained mixture of these lipids forms together with water micelles(Lindmark et al, J. Pharm. Exp. Ther. 275, 958-65, 1995).

Liposomes consists of phospholipids and are formulated by a relativelylengthy and cumbersome process which i.a. involves organic solvents.Furthermore, the phospholipids are expensive.

As described below in the present invention, a similar immunologicalresponse can be obtained only by mixing the antigen with a lipidformulation which contains less complicated lipids having asubstantially lower price and which can be formulated on a commercialbasis in a very simple way.

Another systems that to some extent are similar to the present inventionare formulations based on triglycerides. However, these systems arescientifically defined as emulsions of triglycerides where surfactantsare used for stabilization. As stabilizers phospholipids or any othertype of amphiphilic molecules such as Tween® are normally used.Furthermore, the appearance of such emulsions are normally milky,indicating a size of the oil droplets of about 1 μm. It is well-knownfor the person skilled in the art that these surfactants are excellentadjuvants. Thus, the adjuvant properties of oil emulsions are primarilydue to the characteristics of the surfactant and not of the triglyceridecomposition.

In PCT/DK94/00062 is disclosed a formulation for the topicaladministration of antigens and/or vaccines to mammals via the mucosalmembranes. Said application disclose in the examples that the onlyformulation that enhances the immune response is a combination ofcaprylic/capric acid glycerides with polyoxyethylene sorbitan monoester(Tween 20®).

As exemplified in the present invention it is shown that a combinationbetween a monoglyceride and a fatty acid can stimulate the immune systemto produce antibodies and induce protective immunity. Furthermore thepresent invention shows that the disclosed formulation is able toproduce high antibody titers by parenteral administration.

Thus, it was surprisingly found that the administration of antigensand/or vaccines to an animal either via the mucosal route orparenterally using a formulation comprising monoglycerides and/or fattyacids as a particulate lipid system can improve the immunologicalresponse towards the admixed antigens and/or vaccines. Themonoglycerides are selected from a group with the general formula of1-acyl-glyceride, wherein the number of carbon in the acyl chain may bevaried between 8 and 24, preferably between 12 and 18. The acyl chainmay be either saturated or unsaturated. The concentration of themonoglyceride may be in the range of 0.1-50 g per 100 ml of water,preferably in the range of 1-20 g per 100 ml of water. The fatty acidconcentration may be in the range of 0.1-50 g per 100 ml formulation,preferably in the range of 1-20 g per 100 ml water. When monoglyceridesand fatty acids are formulated together the percent ratio ofmonoglyceride in fatty acid may be varied between 1 to 99%, preferablybetween 10 to 90%.

An enhancement of the immunological response after administration ofmonoglycerides and/or fatty acids together with antigens and/or vaccineshas not been suggested anywhere in the prior art.

The present invention describes that mixtures of antigens with relevantlipids stimulates the body to generate protective immunity. Anotheradvantage of the present invention is the simple formulation process andas compared to entrapment no material (antigen) is lost in the process.As an example can be mentioned that in the process of entrapment inliposomes the recovery is normally 10-20%. The rest is lost in theprocess.

Reports in the literature as discussed above, shows that by mixingliposomes and antigen an immune response is detected afteradministration to the mucosa.

However, the examples in this invention as described below shows thatthe system can be even more simplified by the use of lipids that aremore stable, cheaper and which can be formulated to particles in a moreconvenient and simplified way.

The invention is exemplified by the following examples showing that theprinciple of co-administration of antigens, immune stimulatingsubstances associated or in combination with particles function as anadjuvant.

EXAMPLE 1

A suspension of mono-olein was produced by adding 3 g mono-olein to 50ml of a 0.6% Pluronic-127® solution in phosphate buffered saline pH 7.4,whereafter the mixture was sonicated with a probesonicator for 4minutes. The obtained milky suspension contained particles with amaximal size of about 2 μm as determined by light microscopy.

EXAMPLE 2

A negatively charged micelle suspension of mono-oleate was produced bymixing of 0.5 g of oleic acid with 5 ml of 0.35 M NaOH and sonicatedwith a probesonicator for 5 seconds. Thereafter 3 g mono-olein and 50 ml0.9% NaCl was added whereafter the mixture was probesonicated for 4minutes. The monester content of the mono-oleate was over 95% with aacyl chain containing 92% oleate and 6% linoleic acid. The pH wasadjusted to 8.3. The obtained completely clear homogenous solutioncontained particles with a size of below approximately 0.2 μm asdetermined by visual inspection. It is known that if a clear solution isobtained the particle size is below approximately 0.2 μm, a slightlyopalescent bluish appearance indicated a size of approximately 0.2-0.5μm and if the appearance is milky the size is above approximately 0.8μm.

EXAMPLE 3

A positively charged micelle suspension of mono-olein was produced bymixing 0.5 g lauryl-amine and 3.5 ml of 0.5 M HCl followed by sonicationfor 5 seconds. Thereafter 3 g mono-olein and 50 ml of water was addedwhereafter the mixture was probesonicated for 4 minutes. The pH wasadjusted to between 4 and 5 using 0.5 M HCl. The obtained completelyclear homogenous solution contained particles with a size of belowapproximately 0.2 μm.

EXAMPLE 4

A mixture of particles according to Example 1 and diphtheria toxoid wasadministrated subcutanously to mice followed by a booster after 21 days.After 30 days blood samples were obtained which were assayed for IgGantibodies against diphtheria toxin as well as Neutralization titers(NT) using Vero cells. The serum from Alum (n=5) and monoolein (n=5)groups was pooled and assayed. The mice receiving nasal boost andresponded (=3 of 5) were assayed on an individual basis. In arbitraryunits is shown in Table 1 the IgG titers and neutralization titers Theresults showed that both IgG as well as protective antibody titers wereat the same level as compared to the control group which received themarketed product comprising diphtheria toxoid adsorbed on Alum(Al(PO₄)₃). Also seen is that high IgG titers always were accompanied byhigh neutralization titers indicating that the formulation does notdestroy the antigenic sites that are important for protective immunity.TABLE 1 Dose diphtheria IgG titer NT titer toxoid μg (arb. units) (arb.units) Alum 15 + 15 32000 40000 Alum 3.5 + 3.5 22000 20000 Mono-oleinsuspension 15 + 15 24000 20000 Mono-olein suspension 3.5 + 3.5 3500 5000Nasal boost 7 + 4 45000 10000 Nasal boost 7 + 4 19000 2500 Nasal boost7 + 4 19500 5000

EXAMPLE 5

Particles were prepared according to Example 2 with a finalconcentration of monoglyceride of 200 mM and of fatty acid of 200 min.Diphtheria toxoid (2.9 μl, 4.4 mg/ml) was mixed with 200 μl of themicelle suspension and administrated subcutanously to mice followed by asubcutaneous booster after 21 days. Both the primary and the boosterdose of the toxoid was 10 μg. After 30 days blood samples were obtainedwhich were assayed for IgG antibodies against diphtheria toxin. Theresult showed (Table 2) that the arbitrary IgG titers with respect tothe formulation with mono-olein (MO) and oleic acid (C18:1) were at thesame level as compared to the control group which received the presentmarketed product comprising diphtheria toxoid adsorbed on Alum(Al(PO₄)₃). The other combinations of monoglycerides and fatty acidsgave slightly declining responses which correlated to declining lengthof the acyl chain (M12=lauryl-1-glycerate; M10=capric-1-glycerate;C12=lauric acid; C10=capric acid; C8=caprylic acid). N.D.=Not Done;indicates that there were only five mice in these groups. TABLE 2 IgGresponse of individual mice (n = 5 or 6) after sc/sc administration ofdifferent formulations containing monoglycerides and fatty acids. 1 2 34 5 6 Alum 18200 18200 9600 9600 18200 N.D. MO/C18:1 18200 18200 1820018200 9600 N.D. MO/C8 9600 9600 4800 9600 4800 9600 M12/C12 18200 96004800 18200 9600 18200 M10/C10 4800 110 2400 1200 2400 4800

EXAMPLE 6

The same procedure as in Example 4 with the difference that the boosterdose was given nasally instead of subcutanously. The dose of diphtheriatoxoid was 10 μg both at the primary immunization as well as at thenasal booster administration. In the same experiment a dose-response isdemonstrated that is obtained when tree different amounts of lipid (seeTable 3) was administrated. The arbitrary IgG titer is seen in Table 4.Besides the dose-response effect where lower IgG titers is seen at lowerconcentrations of lipids there is also seen a higher variabilityregarding response in the groups receiving lower doses. This variabilityis not seen at higher dose levels indicating that an adjuvant effect isnot only seen with respect to obtaining high titers but also regardingreduction of the variability of the response. TABLE 3 Amount of lipidsin μmol administrated to mice sc or nasally. Dose level Dose lipid(μmol) sc Dose lipid (μmol) nasally high 40 1.5 medium 4 0.15 low 0.40.015

TABLE 4 IgG titers in individual mice (n = 6) after administration of 2× 10 μg of diphtheria toxoid to mice either sc/sc or sc/nasally. 1 2 3 45 6 MO/C8 sc/sc high 4800 4800 9600 4800 9600 9600 MO/C8 sc/nas high9600 1200 4800 4800 4800 9600 MO/C8 sc/sc medium 4800 1200 9600 24004800 4800 MO/C8 sc/nas medium 2400 600 2400 600 2400 4800 MO/C8 sc/sclow 300 2400 9600 2400 4800 2400 MO/C8 sc/nas low 600 600 1200 150 4800150

EXAMPLE 7

Two different lipid formulation containing mainly medium length acylchains (Composition A) and long acyl chains (Composition B) were tested.The compositions are seen in Table 5. TABLE 5 Monoglyceride Fatty acidComposition A Monooleate 25 mM Caprylic acid 90 mM Monomyristate 25 mMMonolaurate 25 mM Monocaprate 25 mM Composition B Monooleate 200 mMOleic acid 200 mM

The formulations were administrated to mice s.c. or nasally with abooster after three weeks s.c. or nasally. Blood samples were takenafter another week. The arbitrary IgG titers are seen in Table 6.

The results in Table 6 demonstrates that in order to achieve a goodresponse after primary as well as booster administration by the nasalroute Compositions B is to be preferred. TABLE 6 1 2 3 4 5 6 CompositionA 2400 4800 4800 18200 4800 4800 sc/nas Composition A <100 36400 <100<100 300 <100 nas/nas Composition B 18200 18200 36400 18200 <100 N.D.sc/nas Composition B 4800 9600 18200 18200 2400 9600 nas/nas

EXAMPLE 7

A mixture of monoolein (200 mM) and caprylic acid (200 mM) was mixedwith formalin inactivated influenza virus (strain SDA/94) andadministrated s.c. at the first occasion to mice followed by a nasalbooster three weeks later. The dose was 0.05 μg HA and blood sample weretaken 3 weeks after the booster dose and assayed for agglutinationtiters (HI) against HA. The results (Table 7) showed that the HI titersin the group receiving the virus together with the adjuvants was at ahigher level as compared to the group receiving the virus in PBS. TABLE7 HI titers in mice receiving formalin inactivated influenza virus afters.c. primary injection and nasal booster. 1 2 3 4 5 6 PBS N.D. 80 N.D.40 N.D. 80 MO/C8 320 320 640 160 320 *N.D. = not detected* = dead

EXAMPLE 8

Micelles according to Example 2 was mixed with formalin killed rotavirus particles and subsequently administrated to female mice. Afterthree immunizations the mice were made pregnant whereafter the new-bornmice were challenged nasally with live rota virus. The figures indicatethe animals that acquired protection after challenge as compared to thetotal number of animals in that group. The result from this challenge isseen in Table 8. TABLE 8 Protection after challenge of rota virus tobaby mice where the mother was vaccinated with a lipid formulationaccording to the invention. Group Administration Protection Salineim/im/im 2/8 Micelles im/im/im 4/4 Micelles im/nas/nas 6/7

As can be seen from the results there is a good protection both afterthree intramuscular administrations as well as after a primaryintramuscular immunization followed by two nasal administrations.

EXAMPLE 9

To evaluate the toxicity of the lipid formulations these wereadministrated into the rat nasal cavity whereafter the rats were killedand the nasal mucosa were prepared for light, fluorescence as well asscanning electron microscopy (SEM). Formulations according to Example 1and Example 2 were tested. Only the mono-olein/pluronic suspensionshowed minor changes in the mucosal surface using the SEM. No effectscould be detected under light or fluorescence microscopy. The micellescontaining mono-olein and oleic acid were unable to provoke any changesin the mucosal membranes.

EXAMPLE 10

Caco-2 cells, which are a human cell line originating from a coloncancer can be made to grow as a epithelial mono layer. These cells arefrequently used to examine different substances ability to influence thetransport of biological substances through epithelial cells and has in anumber of experimental systems been shown to give a good correlation toin vivo data regarding uptake from the gut into the bloodstream. Asmarker substances for transport through the cells Na-flouresceine ormannitol is used. The experiments with the lipid formulations accordingto this invention showed an enhanced transport through the Caco-2 cellsat non-toxic concentrations.

1-15. (canceled)
 16. A method of immunizing a human or animal, themethod comprising administering to a human or animal by mucosaladministration a vaccine composition comprising an adjuvant componentcomprising: I) monoglyceride; ii) fatty acid; and, iii) water, whereinthe percent weight ratio of monoglyceride and fatty acid is between 10to 90 together with an immunogenic quantity of an antigen componentselected from the group consisting of diphtheria toxoid, influenza androta virus antigen.